Degradation of the ABA co-receptor ABI1 by PUB12/13 U-box E3 ligases

Clade A protein phosphatase 2Cs (PP2Cs) are abscisic acid (ABA) co-receptors that block ABA signalling by inhibiting the downstream protein kinases. ABA signalling is activated after PP2Cs are inhibited by ABA-bound PYR/PYL/RCAR ABA receptors (PYLs) in Arabidopsis. However, whether these PP2Cs are regulated by other factors remains unknown. Here, we report that ABI1 (ABA-INSENSITIVE 1) can interact with the U-box E3 ligases PUB12 and PUB13, but is ubiquitinated only when it interacts with ABA receptors in an in vitro assay. A mutant form of ABI1-1 that is unable to interact with PYLs is more stable than the wild-type protein. Both ABI1 degradation and all tested ABA responses are reduced in pub12 pub13 mutants compared with the wild type. Introducing the abi1-3 loss-of-function mutation into pub12 pub13 mutant recovers the ABA-insensitive phenotypes of the pub12 pub13 mutant. We thus uncover an important regulatory mechanism for regulating ABI1 levels by PUB12 and PUB13.

Immunoblotting analysis with anti-ACTIN antibody. Figure 7. ABI1 protein is more stable in aba2-21 than the wild type in the cell-free 26S proteasome assay. Total proteins were extracted from ABA deficient mutant aba2-21 (contains less than 10% ABA of the wild type) and the wild type. ABI1-His protein purified from E. coli was added together with the extracted proteins into the ATP containing cell-free solution, and incubated for 0, 0.5, 1 and 2 h.

Supplementary
Then the proteins were used for immunoblotting analysis using anti-His antibody.
ACTIN was used as a loading control. Figure 8. ABI1 protein is lower in pyr1 pyl1 pyl2 pyl4 quadruple mutant than in the wild type, but more stabilized in protein solution from pyr1 pyl1 pyl2 pyl4 quadruple mutant than from the wild type.

Supplementary
A. ABI1 protein is accumulated less in pyr1 pyl1 pyl2 pyl4 quadruple mutant than in the wild type in both no ABA treatment and ABA treatment. Total proteins extracted from 7-day-old seedlings treated with 50 M ABA for 6 hr or without ABA were used for immunoblotting analysis with anti-ABI1 antibody. ACTIN was used as a loading control.
B. Comparison of ABI1-His stability between pyr1 pyl1 pyl2 pyl4 quadruple mutant and the wild type in a cell free assay. ABI1-His protein purified from E. coli was added to the ATP containing cell-free solution with total proteins from pyr1 pyl1 pyl2 pyl4 quadruple mutant or the wild type, respectively, in absence (upper) or presence (lower) of MG132 (50 µM). The proteins were used for immunoblotting analysis after cultured for different times. ACTIN was used as a loading control. A. ABI1 protein is higher in abi1-1 (Col) than in the wild type without ABA treatment, but lower with ABA treatment. Proteins extracted from seedlings treated with 50 µM ABA for 6 hr or without ABA treatment were used for immunoblotting analysis with anti-ABI1 antibody. ACTIN was used as a loading control.
B. The degradation of the mutated ABI1 G180D (ABI1-1) is greatly reduced in the cellfree 26S proteasome assay. The protoplasts were transiently expressed with the Pro35S:ABI1-Myc or Pro35S:ABI1-1-Myc plasmid in the wild type (Col). The total extracted proteins were equally divided into four parts, which were incubated in in 26S proteasome degradation buffer for different times. Immunoblotting analysis was carried out with anti-Myc antibody. ACTIN was used as a loading control. Figure 10. pub12 pub13 mutants are more sensitivity to freezing stress than wild type (Col).

Supplementary
Comparison of freezing tolerance of pub12 pub13 mutant and the wild type (Col).
Two-week seedlings grown on MS plates at 22 º C were treated at -5 º C for 1 h (nonacclimation, NA), or first treated at 4 º C for 4 days, and then treated at -9 º C for 1 h (Cold-acclimation, CA). After 3 days at 22 º C, the images were taken (A), and survival rates (B) and ion leakage (C) were measured. Data are means of three replicates ± SD, and the asterisks indicate significant differences compared with the Col under the same treatment conditions (**p < 0.01, t test).

Primers for confirming T-DNA insertions
Primer name sequence